The overall goal of the research project is to provide new information regarding the mechanism of calcification of cartilage, which is an important phenomenon as an integral aspects of calcification have been studied in detail, however the process of chondrocyte maturation has receive surprisingly little attention. The hypothesis to be tested is that chondrocytes exhibit a particular phenotypic expression relating to mineralization which consist of cellular hypertrophy, increase in alkaline phosphates (AP), and release of AP-containing matrix vesicles, in addition to the usual chondrocytic features of proteoglycan synthesis and type Ii collagen synthesis, and that these features ad regulated by growth factors. The proposal is directed toward defining the factors controlling chondrocyte hypertrophy. The specific aims of the proposal are (1) Investigation of growth factors (specifically Transforming growth factor TGF-beta), epidermal growth factor (EGF), and heterotopic bone extract (HBE) for effects on chondrocyte hypertrophy in a conditions which promote chondrocyte hypertrophy and mineralization, such as serum supplements and substrate deprivation; (3) study of the correlation of cell size and AP content, and of possible effects of AP inhibitors on hypertrophy; (4) comparison of growth factors effects on articular versus growth plate chondrocytes; (5) development of centrigual elutriation as a technique for separating chondrocytes in different stages of maturation to study growth factor effects on different cell populations; (6) study of the correlation between hypertrophy, vesiculation, and cytosolic calcium content using florosent calcium-binding dyes and (7) assay of chondrocytes for TFG-beta activity to determine whether TGF-beta may be an autocrine factor controlling chondrocyte maturation. A cell isolation and culture model will be used for these experiments, and hopefully will elucidate the mechanism of chondrocyte hypertrophy. Preliminary data implicate TGF-beta as probably influencing, at lease in cell culture, chondrocyte hypertophy, matrix vesicle biogensis, and expression of cellular AP, all of which are essential events in the mineralization of cartridge.